Acquisition system for slide analysis

ABSTRACT

A closed loop scanning and positioning system for finding and positioning white blood cells includes a multifaceted rotating mirror, an optical system, two photoelectric sensors, two light sources and a logic circuit. The logic circuit produces signals which are used to drive a microscope stage in x and y directions to cause the blood cell to be positioned within a small aperture.

FIPSZIZ OR 3,864,564

750/101 0 United States Patent 11 1 1111 3,864,564 Adkins 1 Feb. 4, 1975 1 ACQUISITION SYSTEM FOR SLIDE 3,515,877 6/1970 Baxter 250/201 ANALYSlS 3,541,338 11/1970 Duda 250/201 [75] Inventor: W1ll1am J. Adklns, Raleigh, N.C. Primary Examiner james W. Lawrence [73] Assignee: Corning Glass Works, Corning, Assistant Examiner-D. C. Nelms N.Y. Attorney, Agent, or Firm-Walter S. Zebrowski; 122 Filed: Sept. 26, 1973 A closed loop scanning and positioning system for 3 0 623 finding and positioning white blood cells includes a J 'f t t I t t 158 Field 61 Search 250/201, 222 PC; 356/39, mu ed "Mung Sys em W0 photoelectric sensors, two light sources and a logic 356/401235/92 318/565 577 circuit. The logic circuit produces signals which are used to drive a microscope stage in x and y directions [56] References (med to cause the blood cell to be positioned within a small UNITED STATES PATENTS aperture.

2,859,653 11/1958 Blackstone... 250/201 3,466,514 9/1969 Brunner 318/577 15 Clams 8 Draw F'gum 2 PULSE SHAPING 56 ACQUlSlTlON smut NORMAL smGE CIRCUITRY L Y POSITIONING MOTION MOTOR 5 PATENIED rm mars BINOCULARS SHEET 10F 5 PULSE SHAPING 56 ACQUISITION SYNCH +x I XPOSITICNING Log C YRETTSSNG PATENIED 3. 864.564

SHEET 2 OF 5 58 YET??? PATENTED SHEET 3 BF 5 mwhznou mmhznou 1 ACQUISITION SYSTEM FOR SLIDE ANALYSIS BACKGROUND OF THE INVENTION This invention relates to a system for converting the optical image of a laboratory slide to electrical signals and more particularly to an acquisition system for bringing a specimen of interest on the slide into position for analysis.

In the analysis of blood samples, the blood is smeared on a laboratory slide and the smear is stained. By counting the leukocytes on the stained smear, laboratory technicians perform what is referred to as a white blood cell differential. Automation of this differential has significant economic impact because the differential is performed so frequently at every hospital. A thesis by J. W. Backus, An Automated Classification of the Peripheral Blood Leukocytes by Means of Digital Image Processing", University of Illinois, Chicago, 1971, describes one automated system.

In a system developed by my co-employees, a scanning unit (in this case a TV. camera) linearly sweeps a vidicon target subjected to intense illumination which passes through the smeared slide. Such a system is described in copending application Ser. No. 353,004, filed Apr. 20, 1973.

In order to count and classify the blood cells on a slide it is necessary to successively find each blood cell and focus its image on the vidicon target. The focusing system is described in copending application Ser. No. 399,619, filed Sept. 21, I973, Amos et al. The system for successively locating and bringing each blood cell into a position for analysis is the subject of the present application.

SUMMARY OF THE INVENTION In accordance with this invention a specimen of interest on an optical slide is positioned with respect to a yiewing aperture by a logic circuit which receives signals from light sensing devices. A rotating mirror successively scans portions of the-optical image of the slide across one of the light sensing devices. The logic circuit produces control signals which are used in positioning of the slide. If no specimen of interest, in this case a blood cell, is detected during a scan, the logic produces a signal which moves the microscope slide in the y direction, orthogonal to the direction of scanning so that another field of view can be scanned.

A second light sensing device receives light at the beginning of each scan of the rotating mirror. This light sensing device produces a synchronizing pulse which is applied to the logic circuit. If a cell is detected during a scan, the logic circuit compares the time occurrence of the acquisition of the cell to the time of the synchronizing pulse. In this manner the logic circuit produces signals which specify the direction in which the slide is to be moved along the x axis in order to center the cell in the viewing aperture.

The foregoing and other objects, features and advantages of the invention will be better understood from the following more detailed description and appended claims.

DESCRIPTION OF THE DRAWINGS FIG. 1 is an optical and electrical schematic diagram of the system of this invention;

FIGS. 2A2D depict the acquisition of a blood cell; and

FIGS. 3A-3C show the electrical schematic of the logic circuit.

DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 shows the optics for a system for scanning and counting leukocytes on a blood smeared slide 10. Light from the lamp 12 passes through lens 14, is reflected from fold mirror 16, and passes through condenser lens 18. The light passes through the slide 10 and is collected by the objective lens 20. A first beam splitter 22 reflects approximaterly 40 percent of the light to the automatic focus and acquisition system of which this invention is a part. The remainder of light is reflected by mirrors 24 and 26 to an optical-to-electrical convertor which scans and analyzes a blood cell whose image is centered on the aperture 28. The light image passing through the aperture 28 is applied to relay lens 30, to optical preprocessor 32 and to the compensator 34. The cell image is applied to vidicon type TV. camera 36 which produces electrical signals which are ultimately converted into digital signals representing the characteristics of each blood cell. This conversion is more fully described in the aforementioned Cotter application.

A portion of the focused light from beam splitter 22 strikes a rotating multifaceted mirror 38. Light striking mirror 38 forms an image of the slide which is parfocal with the image applied to the converter. The rotating mirror reflects a portion of the slide image onto a light sensing device 40. Light sensing devices 42 and 44 are used in the focusing system as more fully described in the copending Amos et al application. A very narrow slice of the slide image is scanned as the mirror 38 rotates. The light sensing device 40 produces an output proportional to the light absorption of the area of the slide being scanned. This signal is amplified and peak detected in the pulse shaping circuits 46. This circuit produces acquisition pulses indicating the detection of a blood cell on a slide only for signals above a given threshold.

A second light source 48 is focused on the rotating mirror 38 and the focused light is swept across a second light sensing device such as photocell 50. The light sensing device 50 is adjusted so that the light from source 48 strikes it at a predetermined position in the scan. In this case, the light strikes photocell 50 in time relation to the beginning of a scan of the mirror across the slide. The photocell 50 produces a synchronizing signal to mark the beginning of the sweep by a given mirror facet.

The present invention is directed to the system which centers an image of each blood cell on the slide onto the aperture 28. The acquisition pulses and synchronizing pulses are applied to logic circuitry 52. The logic circuitry utilizes these pulses to determine if a specimen of interest is in the field of view of one of the scanning segments. If so, the circuitry determines which way the stage must move in order to center the specimen in the aperture. As each facet of the rotating mirror sweeps the field, one of four signals is generated by the logic circuitry. If no specimen is encountered, a normal stage motion signal is generated. This causes the y positioning motor 54 to move the slide one step in the y direction so that another field of view can be scanned. This one step per mirror facet motion continues until a specimen is encountered by the acquisition sensor 40. At this time a directional signal is produced. The directional signal indicates whether the specimen is to be moved to the right or left, i x direction, in order to center the cell on the aperture. The x positioning motor 56 moves the slide in either the +x or the x direction. This motion continues until the logic circuitry produces a cell-centered signal. At this time all slide motion is halted, the cell specimen is centered in the aperture 28, and the vidicon 36 is enabled to allow it to make an analysis of the cell. The stage motors 54 and 56 are stepping motors which move the stage a predetermined distance for each applied pulse.

In the overall system of which this invention is a part, a small digital computer is used in the analysis and storage of information regarding the blood cells. It is convenient to use this digital computer for control purposes. Therefore, it will be preferred to apply the normal stage motion, directional and cell-centered signals from the logic circuitry to the digital computer. However, for purposes of explanation it is clearer to describe the logic circuitry as controlling the x and y positioning motors 54 and 56 and the vidicon 36. The modifications required to use the digital computer for control are well understood by those skilled in using these devices.

The operation can best be explained with reference to FIGS. 2A-2D. These figures depict the image of a blood smeared slide. Included in this image is a specimen of interest, notably the blood cell 58. The figures depict the image in relation to the aperture 28 and the figures depict the image in relation to the field of view which is scanned and which is shown as the shaded areas. (It should be remembered that the image applied to aperture 28 is parfocal with the image scanned by the rotating mirror 38. Therefore, it is accurate to depict the scan field of view as the shaded area in relation to the aperture 28 in this manner.)

A first scan by one facet of the mirror 38 is depicted in FIG. 2A. During this scan no blood cell is detected by the acquisition detector 40. Therefore, the logic circuitry produces a normal stage motion signal which moves the slide by one increment in the y direction.

" During the next scan depicted in FIG. 2B, the acquisition detector 40 does not produce an acquisition pulse. Again a normal stage motion signal moves the slide one increment in the ydirection to the position depicted in FIG. 2C. During this scan the acquisition detector 40 produces a pulse indicating that a blood cell lies in the field of view of this scan. Further, the logic circuitry detects that the cell lies to the left of the aperture 28. Therefore, the logic circuitry produces a directional signal which moves the slide in the +x direction. This directional signal is produced until the slide is positioned with the cell 58 centered on the aperture 28 as depicted in FIG. 2D.

The logic circuitry for producing signals in this manner is shown in FIGS. 3A-3C. A synchronizing pulse from the synchronizing detector 50 is applied to set the flip-flop 60 at the beginning of each scan by a mirror facet. If there are more than one sync pulse during a scan, due to noise, only the first sync pulse sets the flipflop 60. The flip-flop 60 triggers the one-shot multivibrator 62. By adjusting the monostable time period of multivibrator 62, it is possible to center the aperture with respect to the viewing binoculars used by the operator.

One-shot multivibrator 62 triggers the one-shot multivibrator 64 which produces a pulse of predetermined width. This is used to set the flip-flop 66. When the flipflop 66 is set an oscillator 68 is turned on. This triggers clock pulses which are applied to one-shot multivibrator 70 which produces pulses ofa predetermined width. These pulses are applied to the binary counters 72 and 74. The outputs of the counters 72 and 74 are decoded by the decoding gates 76 and 78. The decoding circuitry divides each scan line into 60 equal increments. A center pulse is produced at the middle of the scan and an end pulse is produced at the end of the scan. in order to produce the center pulse, the decoding gate 76 has inputs from counters 72 and 74 such that the output of gate 76 goes up when the counters receive the twenty-ninth clock pulse. The thirtieth clock pulse passes through the gate 80. This is the center pulse.

The decoding gate 78 has inputs from counters 72 and 74 such that the output of gate 78 goes up on the fifty-ninth clock pulse. The sixtieth clock pulse passes through the gate 82. This is the end pulse. lt resets flipflops 60 and 66. This cuts off the oscillator. The end pulse also provides a reset for the counters 72 and 74.

If an acquisition pulse occurs during a scan, it passes through AND gate 84 and inverter 86 to set the acquisition flip-flop 88. If an acquisition pulse occurs at a time which coincides with the center pulse, the center pulse passes through AND gate 94 which is enabled by the acquisition pulse from gate 84. The output of gate 94 passes through inverter 96 to set the cell-centered flipflop 98. Flip-flop 98 enables the AND gate 100 and the end pulse passes through it to produce the cellcentered pulse.

If an acquisition pulse occurs during a scan, but it does not coincide with the center pulse, a directional signal is produced which indicates the direction of movement along the scan required to center the acquisition pulse. Whether the acquisition pulse occurs before or after the center pulse is detected by a shift register which includes a first stage flip-flop 102 and a second stage flip-flop 104. The first stage flip-flop is initially set; it is reset by the center pulse. Upon the occurrence of the acquisition pulse, the state of the first stage flip-flop 102 is set into the second stage flip-flop 104. If the acquisition pulse occurs before the center pulse, a set state is transferred from the flip-flop 102 to flipflop 104. lfthe acquisition pulse occurs after the center pulse, a reset condition is transferred from flip-flop 102 to flipflop 104. If an acquisition has occurred, the end pulse will pass through AND gate 106 and interrogate the AND gates 108 and 110. If the acquisition occurs before the center pulse, the flip-flop 104 is set so that the connection from flip-flop 104 to the gate 110 is high. This allows the pulse from gate 106 to pass through gate 110 to produce a directional signal indicating a move in the +x direction. Conversely, if the acquisition pulse occurs after the center pulse, the flipflop 104 is reset and the inverter 112 applies a high condition to the gate 108. The pulse from gate 106 passes through the gate 108 to produce a directional signal indicating a move in the x direction. If there is no acquisition pulse during a scan, the acquisition and cell-centered flip-flops 88 and 98 will still be reset at the time of the end pulse. The bottom outputs of these flip-flpos acts through OR gate 90 to enable the AND gate 92. The AND gate 92 passes the end pulse to produce a normal stage motion signal.

Priority circuitry is connected so that only one of the cell-centered, directional, and normal stage motion signals can be produced to the exclusion of the others. With the circuitry described thus far, it would be conceivable that all three signals would be produced at the end of a given scan. Of course it is necessary that only one produced so as to avoid confusion. The highest priority signal is the cell-centered signal. When the flipflop 98 is set, the lower output from this flip-flop is low. This acts through the gate 114 to hold the acquisition flip-flop 88 in the reset condition. This prevents an end pulse from passing through AND gate 106 to produce a directional signal. The bottom output of flip-flop 98, which is low because the flip-flop is set, acts through OR gate 90, to block AND gate 92. This prevents the production of a normal stage motion signal.

The second priority is the production of directional signals. If the acquisition flip-flop 88 is set, the lower output of the flip-flop is applied through OR gate 90 to block the gate 92. No normal stage motion pulse can be produced.

The only time a normal stage motion pulse is produced is at the end of a scan in which neither the acquisition flip-flop 88 nor the cell-centered flip-flop 98 has been set. ln this case, the end pulse can pass. through both gates 90 and 92 and produce a normal stage motion pulse.

Refer once more to the adjustment for the one-shot multivibrator 62. This manual adjustment moves the center pulse in relation to the sync pulse. It is desirable that the center pulse occur with respect to the scan at a point which is approximately in the middle of the field of view of the camera 36. The one-shot multivibrator 62 can be adjusted until this condition is met. Similarly, the frequency adjustment of the oscillator 68 can be adjusted. By lowering the frequency of these pulses, the si tty clock pulses will encompass a greater length of scan. Similarly, increasing the frequency decreases the length of scan.

While a particular embodiment of the invention has been shown and described, various modifications are within the true spirit and scope of this invention. The

' appended claims are, therefore, intended to cover all such modifications.

What is claimed is: 1. In a system producing an electrical output representing the optical characteristics of an analytical slide containing a specimen of interest, including:

conversion means producing said electrical output representing the optical characteristics of said slide,

means for projecting an optical image of said analytical slide to said conversion means,

a light sensing device,

means for successively scanning a portion of said optical image, said light sensing device producing an output representing the light absorption of the area of the slide being scanned,

a pulse shaping means connected to the output of said light sensing device and producing an acquisition signal when the area of the slide being scanned contains a specimen of interest, and

a positioning mechanism for positioning said slide, an

acquisition system comprising:

6 synchronizing means producing a synchronizing signal at a predetermined point in each scan, and logic circuitry responsive to said synchronizing signal and to said acquisition signal for producing control 5 signals, said control signals being used to control said positioning mechanism to position said slide so that the image of a specimen of interest is centered on said conversion means. 2. The system recited in claim I wherein said syn- 10 chronizing means comprises:

a rotating mirror, a light source focused on said rotating mirror, and a second light sensing device, said second light sensing device and said light source being positioned so that reflected light striking said second light sensing device is in time relation to the beginning of a scan of said mirror across said slide, said second light sensing device producing a synchronizing signal upon reception of said light.

3. The system recited in claim 1 wherein said logic circuitry includes means for producing a normal stage motion signal if no specimen is detected during a scan said normal stage motion signal being applied to said positioning mechanism to move said slide by an increment in a direction orthogonal to the direction of scan. 4. The system recited in claim 1 wherein said logic circuitry includes:

means responsive to said synchronizing signal for producing a center pulse occurring approximately in the middle of each scan, and means responsive to the occurrence of an acquisition signal before or after said center pulse for producing directional signals representing the direction of motion of said slide along the direction of scanning required to center said specimen on an aperture of said conversion means, said directional signals being applied to control said mechanism. 5. The system recited in claim 4 wherein said logic circuitry further comprises:

means for producing a specimen-centered signal when said acquisition pulse coincides with said center pulse thereby indicating that said specimen is centered on said aperture.

6. The system recited in claim 4 wherein said means for producing a center pulse comprises:

an oscillator producing clock pulses, a pulse counter, said clock pulses being applied to said pulse counter upon the occurrence of said synchronizing pulse, and

a decoder, the outputs of said counter being applied to said decoder, said decoder producing said center pulse upon the occurrence of a predetermined count in said counter.

7. The system recited in claim 4 wherein said means for producing directional signals comprises:

a shift register having a first stage which is set by said synchronizing signal and which is reset by said center pulse and a second stage which is switched by said acquisition signal, said first stage being con nected to said second stage so that the state of said first stage is set into said second stage when said acquisition signal occurs, the output of said second stage being a bistable directional signal which indicates whether said acquisition signal occured before or after said center pulse.

8. The system recited in claim 6 further comprising:

a second decoder, the outputs of said counter being applied to said second decoder to produce an end pulse coincident with the end of each scan,

a specimen-centered flip-flop which is set by the coincidence of said acquisition signal and said center pulse, and

a gate, said end pulse and the output of said specimen-centered flip-flop being applied to said gate, said gate producing a specimen-centered signal which is applied to said conversion means at the end of a scan so that said conversion means converts the optical image ofa specimen which is centered on said aperture into an electrical output.

9. The system recited in claim 8 further comprising:

an acquisition flip-flop, said acquisition flip-flop being set by said acquisition signal, and

a second gate, said end pulse and the output of said acquisition flip-flop being applied to said second gate to produce a directional signal, said directional signal being applied to said positioning mechanism to move said slide along the direction of scanning required to center said specimen on said aperture.

10. The system recited in claim 9 further comprising:

means for producing a normal stage motion signal if no specimen is detected during a scan, said normal stage motion signal being applied to said positioning mechanism to move said slide by an increment in a direction orthogonal to the direction of scan, and

priority circuitry connected to enable said logic circuitry to produce only one of said specimen- .centered, directional and normal stage motion signals to the exclusion of others in the order of priority of, a specimen-centered signal, a directional signal, and a normal stage motion signal.

11. The system recited in claim 6 wherein said oscillator has an adjustable frequency to adjust the length of each scan.

12. The system recited in claim 6 comprising:

an adjustable one-shot multivibrator, said synchronizing pulse being applied to trigger said one-shot multivibrator, the output of said one-shot multivibrator being applied to said oscillator to start the production of said clock pulses, said one-shot multivibrator having an adjustable monostable time period so that said center pulse occurs at a position in said scan which coincides with the center of the field of view of said conversion means.

13. The system recited in claim 1 wherein said means for projecting includes a beam splitter which projects one slide image to said conversion means and another slide image to said rotating mirror.

14. The system recited in claim 1 wherein said conversion means includes an aperture, said positioning mechanism being controlled to center the image of a specimen of interest on said aperture.

15. The system recited in claim 4 wherein said synchronizing means comprises:

a rotating mirror,

a light source focused on said rotating mirror, and

a second light sensing device, said second light sensing device and said light source being positioned so that reflected light striking said second light sensing device is in time relation to the beginning of a scan of said mirror across said slide, said second light sensing device producing a synchronizing signal upon reception of said light. 

1. In a system producing an electrical output representing the optical characteristics of an analytical slide containing a specimen of interest, including: conversion means producing said electrical output representing the optical characteristics of said slide, means for projecting an optical image of said analytical slide to said conversion means, a light sensing device, means for successively scanning a portion of said optical image, said light sensing device producing an output representing the light absorption of the area of the slide being scanned, a pulse shaping means connected to the output of said light sensing device and producing an acquisition signal when the area of the slide being scanned contains a specimen of interest, and a positioning mechanism for positioning said slide, an acquisition system comprising: synchronizing means producing a synchronizing signal at a predetermined point in each scan, and logic circuitry responsive to said synchronizing signal and to said acquisition signal for producing control signals, said control signals being used to control said positioning mechanism to position said slide so that the image of a specimen of interest is centered on said conversion means.
 2. The system recited in claim 1 wherein said synchronizing means comprises: a rotating mirror, a light source focused on said rotating mirror, and a second light sensing device, said second light sensing device and said light source being positioned so that reflected light striking said second light sensing device is in time relation to the beginning of a scan of said mirror across said slide, said second light sensing device producing a synchronizing signal upon reception of said light.
 3. The system recited in claim 1 wherein said logic circuitry includes means for producing a normal stage motion signal if no specimen is detected during a scan said normal stage motion signal being applied to said positioning mechanism to move said slide by an increment in a direction orthogonal to the direction of scan.
 4. The system recited in claim 1 wherein said logic circuitry includes: means responsive to said synchronizing signal for producing a center pulse occurring approximately in the middle of each scan, and means responsive to the occurrence of an acquisition signal before or after said center pulse for producing directional signals representing the direction of motion of said slide along the direction of scanning required to center said specimen on an aperture of said conversion means, said directional signals being applied to control said mechanism.
 5. The system recited in claim 4 wherein said logic circuitry further comprises: means for producing a specimen-centered signal when said acquisition pulse cOincides with said center pulse thereby indicating that said specimen is centered on said aperture.
 6. The system recited in claim 4 wherein said means for producing a center pulse comprises: an oscillator producing clock pulses, a pulse counter, said clock pulses being applied to said pulse counter upon the occurrence of said synchronizing pulse, and a decoder, the outputs of said counter being applied to said decoder, said decoder producing said center pulse upon the occurrence of a predetermined count in said counter.
 7. The system recited in claim 4 wherein said means for producing directional signals comprises: a shift register having a first stage which is set by said synchronizing signal and which is reset by said center pulse and a second stage which is switched by said acquisition signal, said first stage being connected to said second stage so that the state of said first stage is set into said second stage when said acquisition signal occurs, the output of said second stage being a bistable directional signal which indicates whether said acquisition signal occured before or after said center pulse.
 8. The system recited in claim 6 further comprising: a second decoder, the outputs of said counter being applied to said second decoder to produce an end pulse coincident with the end of each scan, a specimen-centered flip-flop which is set by the coincidence of said acquisition signal and said center pulse, and a gate, said end pulse and the output of said specimen-centered flip-flop being applied to said gate, said gate producing a specimen-centered signal which is applied to said conversion means at the end of a scan so that said conversion means converts the optical image of a specimen which is centered on said aperture into an electrical output.
 9. The system recited in claim 8 further comprising: an acquisition flip-flop, said acquisition flip-flop being set by said acquisition signal, and a second gate, said end pulse and the output of said acquisition flip-flop being applied to said second gate to produce a directional signal, said directional signal being applied to said positioning mechanism to move said slide along the direction of scanning required to center said specimen on said aperture.
 10. The system recited in claim 9 further comprising: means for producing a normal stage motion signal if no specimen is detected during a scan, said normal stage motion signal being applied to said positioning mechanism to move said slide by an increment in a direction orthogonal to the direction of scan, and priority circuitry connected to enable said logic circuitry to produce only one of said specimen-centered, directional and normal stage motion signals to the exclusion of others in the order of priority of, a specimen-centered signal, a directional signal, and a normal stage motion signal.
 11. The system recited in claim 6 wherein said oscillator has an adjustable frequency to adjust the length of each scan.
 12. The system recited in claim 6 comprising: an adjustable one-shot multivibrator, said synchronizing pulse being applied to trigger said one-shot multivibrator, the output of said one-shot multivibrator being applied to said oscillator to start the production of said clock pulses, said one-shot multivibrator having an adjustable monostable time period so that said center pulse occurs at a position in said scan which coincides with the center of the field of view of said conversion means.
 13. The system recited in claim 1 wherein said means for projecting includes a beam splitter which projects one slide image to said conversion means and another slide image to said rotating mirror.
 14. The system recited in claim 1 wherein said conversion means includes an aperture, said positioning mechanism being controlled to center the image of a specimen of interest on said aperture.
 15. The system recited in claim 4 wherein said synchronizing means comprises: a rotating mirror, a light source focused on said rotating mirror, and a second light sensing device, said second light sensing device and said light source being positioned so that reflected light striking said second light sensing device is in time relation to the beginning of a scan of said mirror across said slide, said second light sensing device producing a synchronizing signal upon reception of said light. 